Genetic Variability of mtdna Sequences in Chinese Native Chicken Breeds

903 Genetic Vribility of mtdna Sequences in Chinese Ntive Chicken Breeds Z. G. Liu*, C. Z. Lei 1, J. Luo 1, C. Ding, G. H. Chen 2, H. Chng 2, K. H. Wng, X. X. Liu, X. Y. Zhng X. J. Xio 2 nd S. L. Wu 2 Poultry Institute, Chinese Acdemy of Agriculturl Sciences, Yngzhou, Jingsu 225003, P. R. Chin ABSTRACT : The vribility of mtdna hypervrible segment I (HVS I) sequences ws investigted in totl of 48 birds belonging to 12 Chinese ntive breeds. Sixteen hplotypes were identified from 35 polymorphic nucleotide sites which ccounted for 6.4% of sequenced 544 bp frgment. Diversity nlysis of the hplotypes showed tht Tibetn, Lngshn nd Henn cockfight hd only one hplotype, while ncient hplotypes existed in Tihe silky nd Chhu. Phylogenetic nlysis of the hplotypes suggested tht Chinese ntive breeds shred 5 mternl lineges nd some breeds would shre the sme mternl linege, regrdless of their externl fetures nd ecologicl types. Both divergent nd phylogenetic nlysis of the hplotypes indicted the close genetic reltionships between the Chinese ntive breeds nd G. g. gllus nd G. g. spdiceus from different res, which implied tht G. g. gllus nd G. g. spdiceus were the originl ncestors of the Chinese ntive breeds. (Asin-Aust. J. Anim. Sci. 2004. Vol 17, No. 7 : 903-909) Key Words : Chinese, Ntive Chicken, Jungle Fowl, mtdna, Hplotype, Originl Ancestor INTRODUCTION Domestic txonomiclly belongs to Glliformes, Phrsinide, Gllus. Chicken breeds re considered to hve originted either from G. gllus or from G. sonnerti (grey jungle fowl), G. lfyettei (Ceylonese jungle fowl) or G. vrius (green jungle fowl), respectively (Mson, 1987). Domestic is the erliest domesticted fowl nd there re 60 ntive breeds in Chin (Chng, 1995). At present, reserchers hve nlyzed the genetic chrcteristics of Chinese ntive breeds using methods such s cytogenetics, biochemicl genetics nd DNA fingerprinting (Zeng, 1987; Cheng et l., 1991; Wng et l., 2003). Mitochondri, one of the importnt orgnelles in eukryotic cells, re presumed to represent bcteri-like orgnisms incorported into eukryotic cells over 700 million yers go (perhps even s fr s 1.5 billion yers go) nd function s the sites of energy metbolism. In higher vertebrtes, mitochondri strictly follow the pth of mternl trnsmission, i.e., the descendents of the sme mternl ncestor hve lmost identicl mitochondri. Mitochondril DNA (mtdna) is the genetic mteril in mitochondrion. Generlly, evolution of mtdna occurred primrily s single bse pir substitutions, with only infrequent mjor sequence rerrngements. Moreover, the * Corresponding Author: Liu, Zhng-guo. Tel: +86-514-7233488, Fx: +86-514-7254416, E-mil: Liuzg007@163.com 1 College of Animl Science nd Technology, Northwest Sci-Tech University of Agriculture nd Forestry, Yngling, Shnxi 712100, P. R. Chin. 2 College of Animl Science nd Technology, Yngzhou University, Yngzhou, Jingsu 225001, P. R. Chin. Received October 31, 2003; Accepted Mrch 22, 2004 rte of mtdna evolution ws bout 5 to 10 times fster thn nucler DNA, nd its genes did not recombine. So mtdna nlysis hs been used to investigte the genetic bckgrounds of both closely relted species nd individuls within species. The D-loop region of mtdna is known to be more vrible in sequence thn in other regions nd thus hs been frequently used by geneticists for phylogenetic nlysis of closely relted breeds within species. Phylogenetic reltionships mong Chinese ntive breeds hd been determined before using RFLP of mtdna (Wng et l., 1994; Zhou et l., 1997). However, results of these studies provided only limited informtion. Sequencing of mtdna llowed for more powerful phylogenetic inference becuse it cn detect ll the polymorphic sites present. Fu et l. (2001) were the first to use D-loop sequence polymorphism to determine phylogenetic reltionships mong 5 ntive breeds in Zhejing province. In our study, we used the D-loop hypervrible segment (HVS ) of mtdna to determine the genetic differentition bckgrounds nd to probe into the origin of Chinese ntive breeds. MATERIALS AND METHODS Specimen collection Bsed on relted documents (Chng, 1995; Qiu et l., 1988; Compiltion Committee of Annls of Domestic Animl nd Poultry Breeds in Yunnn Province; Guizhou Provincil Development sttion of Animl Breeds, 1997; Li et l., 2001), informtion on the ntive breeds we exmined re listed in Tble 1. We typiclly collected 12 reltively ncient ntive breeds from different niml husbndry culture res in Chin. Ech breed ws represented by 4 specimens from four different lines.

904 LIU ET AL. Tble 1. List of ntive breeds exmined Breed Breed Animl husbndry Time of breed Ecologicl type Collection site Primry loction code culture re origin Tibetn ZJ Other Tibet Qing Zng re b 1,000 yers go Qingyun blotted QY Met Qingyun county, Min Yue re c 900 yers go Gungdong Henn cockfight DJ Recretion Kifeng city, Henn Southest of Chin 2,000 yers go Chhu CH other Dehong county, Southwest of Chin Mny yers go Yunnn Big bone DG Met nd egg concurrent Zhunghe county, Northest of Chin 200 yers go Lioning Beijing youkei BY Met nd egg concurrent An ding re, Beijing North of Chin 250 yers go Lngshn RS Met nd egg concurrent Rudong county, Southest of Chin Very ncient breed Jingsu Yugn Wugu YG Medicine Yugn breed frm Yugn county, Jingxi Xing er Gn re d Hn dynsty (2,000 yers go) Sougung SG Met nd egg concurrent Cilun breed frm Sougung city, Shndong North of Chin 1,500 yers go Tihe Silky SY Medicine Tihe breed frm Tihe county, Jingxi Xing er Gn re d 1,000 yers go Wumeng Wugu WM Medicine Bijie city, Guizhou Bijie city, Guizhou Southwest of Chin Mny yers go Ynjing Wugu YJ Medicine Ynjing county, Yunnn Ynjing county, Yunnn Southwest of Chin Hn dynsty (2,000 yers go) represents Poultry Institute, Chinese Acdemy of Agri. Sci., b contins Qinghi, Tibet nd djcent res. c contins Fujin, Gungdong nd djcent res, d contins Hunn, Hubei, Jingxi nd djcent res. DNA extrction The blood specimens were preserved by 75% ethnol t the volume rte of 1:4 (blood:ethnol) nd stored t room temperture. DNA ws extrcted from these specimens using phenol/chloroform. PCR mplifiction The primers used were those of Dejrdins et l. (1990) nd Rndi et l. (1998) which mplified 544 bp frgment between sites 16,750 nd 543 (GenBnk NC-001323), the forwrd primer ws L 16750 : 5 -AGGACACGGCTTGAAA AGC-3, nd the reverse primer ws H 543 : 5 -ATGTGCC TGACCGAGGAACCAG-3. The regent kits used for PCR were from Shnghi Biologic Engineering Compny. The PCR rection ws crried out in totl volume of 50 µl, nd the finl concentrtion or content of ech component ws s follows, 1 PCR buffer, 2.5 mm MgCl 2, 0.2 mm of ech dntp, 1.7 U DNA polymerse, 0.3 µm of ech primer nd 150 ng DNA templte. PCR ws performed in Progene therml cycler (PE 9600). The rection profiles included n initil denturtion t 95 C for 3 min, followed by 33 cycles, ech consisting of 30 sec denturtion t 94 C, 36 sec primer nneling t 63 C, 56 sec extension t 72 C, nd then finl 5 min extension t 72 C. The mplified products were ll electrophoresed by 2.0% (wt/vol) grose gel in 1 TBE buffer, with 5 v/cm voltge for 1 h. After the run, the gel ws stined with ethidium bromide. ccording to the mnufcturer s instructions. Sequencing ws performed by using n ABI model 377 utomted sequencer (PE). The sequencing primers were the forwrd primer L16750 nd the reverse primer H543. A consensus sequence of pproximtely 544 bp for ech bird resulted from ssembling of sequence reds in both strnds. Dt nlysis All mtdna nucleotide sequences obtined in this work were ligned by using the Clustl X softwre (Thompson et l., 1997), nd identicl sequences were considered s the sme hplotype. Referring to the D-loop sequences of severl breeds in GenBnk, hplotypes dissimilr to those found in the present study were selected to enrich our dt. Using MEGA softwre (Kumr et l., 1993), Kimur 2-prmeter distnce mtrix of ll hplotypes were clculted to construct the unrooted NJ phylogenetic tree with G. lfyettei nd G. vrius s outgroups. Bootstrp confidence levels (BCL) of the phylogenetic tree were estimted by 1,000 rndom bootstrp resmpling of the dt. The hplotypes of the D-loop sequences for ll of the species of Gllus in GenBnk were used to drw the unrooted NJ cldogrm of hplotypes for both Chinese ntive breeds nd Gllus. Bootstrp confidence levels were evluted by 1,000 rndom bootstrp resmpling of the dt. RESULTS PCR products sequencing The mplified products were purified by using the Wizrd TM PCR Preps DNA purifiction kit (Promeg) Sequence nd genetic vrition of mtdna D-loop The sequences we studied hve submitted to GenBnk (from AY465960 to AY466007). The nucleotide

GENETIC VARIABILITY OF mtdna SEQUENCES IN CHINESE NATIVE CHICKEN BREEDS 905 Tble 2. Hplotypes of studied breeds obtined from GenBnk Breed Code of hplotype Accession No. in GenBnk Author Collection site Chhu CH5-6 AF512085, AF512089 Y. P. Liu, et l. Yunnn, Chin Ynjing Wugu YJ5-7 AF512324, AF512326, AF512327 Y. P. Liu, et l. Yunnn, Chin Qinyun blotted QY5 AF512260 Y. P. Liu, et l. Gungdong, Chin Gushi GuS5-8 AF512144, AF512145, AF512146, AF512150 Y. P. Liu, et l. Henn, Chin Bi er yellow BeH5 AF128322 Y. Fu et l. Jingxi, Chin G. g. spediceus spdiceus1-5 AF512182, AF512185, AF512186, AF512187, Y. P. Liu et l. Mynmr AF512188 spdiceus6 AF512174 Y. P. Liu et l. Yunnn, Chin spdiceus7 AB009442 Miyke, T. Los spdiceus8-9 AB009443, D82907 Miyke, T. Thilnd G. g. gllus gllus1-5 AB007720, AB007725, AB007752, AB007756, Miyke, T. Unknown AB007757 gllus6-7 AB009440, AB009439 Miyke, T. Sumtr gllus8-9 AB009438, AB009437 Miyke, T. Lombok gllus10-11 AB009435, AB009434 Miyke, T. Vietnm gllus12 AB009433 Miyke, T. Philippines gllus13 AB009432 Miyke, T. Thilnd G. g. bnkiv bnkiv1-2 AB009430, AB009431 Miyke, T. Indonesi bnkiv3 AB007718 Miyke, T. Unknown G. sonnerti sonnerti D82911 A. Fumihito, et l. Indi G. lfyettei lfyette1-2 D66893, D82910 A. Fumihito. et l. Sri Lnk G. vrius vrius1-2 D82913, D82915 A. Fumihito. et l. Indonesi Tble 3. Nucleotide substitutions in D-loop HVS I of Chinese ntive fowl Substitution Polymorphic sites A/T 268 th, 381 st. C/A 289 th, 316 th, 454 th, 479 th. G/A 232 nd, 259 th, 262 nd, 301 st, 362 nd, 456 th. T/C 153 rd, 187 th, 219 th, 237 th, 245 th, 263 rd, 266 th, 276 th, 281 th, 289 th, 316 th, 322 nd, 330 th, 333 rd, 335 th, 342 nd, 375 th, 383 rd, 387 th, 414 th, 416 th, 467 th, 501 st, 505 th, 534 th. Tble 4. Hplotypes shred mong Chinese ntive breeds Code of Code of Breeds hplotype hplotype Breeds ZJ1 ZJ, CH. YG2 YG, CH, GuS. SG2 SG, SY. BY1 BY, CH, SY, YJ. YJ4 YJ, SY. QY1 QY, CH, DJ, DG, BY, SG, SY, SG4 SG, CH, GuS. YG, WM, RS, BeH. substitutions found in the mtdna D-loop region of Chinese domestic fowl re shown in Tble 3. Anlysis showed tht the lengths of ll the sequences we studied were 5 bses (TACCT t 3 end, not including the primers) longer thn those sequences mplified before by the sme primers (Fu et l., 2001; Liu et l., see Tble 2). When we ligned our sequences with some D-loop sequences of G. g. bnkiv (AB009430 nd AB009431) nd G. g. gllus (AB007720 nd AB007725), the lengths of which were ll more thn 600 bp, the results showed tht the 5 bses (TACCT) should be t the 3 end. Therefore, it is believed tht the sequences in Chinese ntive breeds mplified by the primers in this study should be 544 bp rther thn 539 bp (not including the primers). The 48 smples represented 16 hplotypes of the D-loop hypervrible region. These smples ltogether showed 35 vrible sites of bse substitution. No deletion or insertion ws observed. The verge percentge of polymorphic sites ws 6.4% for the 544 bp region of D-loop HVS I, but the verge percentge of polymorphic sites of ntive breeds in Zhejing province ws just bout 4.45% (Fu et l., 2001). This might be scribed to the differences of smpling res. 11 hplotypes of some relted breeds were identified nd chosen from GenBnk (Tble 2). Among these breeds, Bi er Yellow (BeH) ws n egg type breed while Gushi (GuS) ws reltively ncient breed from Henn province tht ws prt of the originl domestiction re of Chinese ntive. There re 27 different hplotypes from 14 breeds in the study. The results showed tht most of these breeds hd more thn one hplotype, but Tibetn (ZJ), Henn cockfighting (DJ) nd Lngshn (LS) hd only one hplotype ech. On the other hnd, some of these 27 hplotypes were found in more thn one breed (Tble 4).

906 LIU ET AL. 0.20 0.15 0.10 0.05 0.00 QY1** BeH SG2** GuS8 CH6 YJ6 CH2 CH5 WM1 GuS6 BY2 SG4** BY1** YG3 ZJ1** DG2 YJ4** QY5 GuS5 YG1 QY4 YG2** YJ5 YJ1 YJ2 YJ7 GuS7 vrius1 lfyett1 Figure 1. Unrooted NJ phylogenetic tree in Chinese ntive breeds. Hplotypes in the figure with ** ment they were found in more thn one breed. Phylogenetic nlysis in Chinese ntive breeds An unrooted NJ phylogenetic tree of hplotypes in Chinese ntive breeds ws constructed (Figure 1). As whole, these 27 hplotypes were plced into 5 clusters. However the hplotypes of ntive fowls in Zhejing province were grouped into only 2 clusters (Fu et l., 2001). Such clustering disprity might be cused by differences in smpling res. Moreover, Chinese ntive breeds fell into 4 clusters bsed on blood-groups nd blood protein polymorphisms (Cheng et l., 1991) nd microstellite nlysis (Wng et l., 2003). The differences in the genetic mrkers used in the studies might ffect the clustering results s well. Phylogenetic reltionship of Chinese ntive fowl nd other species of Gllus Some hplotypes of other species of Gllus were selected from GenBnk (Tble 2). The verge divergence of hplotypes found in Chinese ntive fowl nd other species of Gllus (Tble 5) showed tht the men divergence indices between Chinese ntive nd G. g. gllus or G. g. spediceus were much lower thn those between Chinese ntive fowl nd G. g. bnkiv or other species of Gllus, which indicted tht Chinese ntive ws more geneticlly close to G. g. gllus nd G. g. spdiceus. Tble 5. Also showed tht, s fr s G. lfyettei, G. sonnerti nd G. vrius, the genetic divergence between G. lfyettei nd G. sonnerti ws reltively lower, which ws similr to the result of Hshiguchi et l. (1993). The phylogenetic tree of hplotypes in Chinese ntive breeds nd other Gllus (Figure 2) lso showed tht, except tht G. g. gllus from Sumtr Islnd (gllus6 nd gllus7) ws slightly remote from Chinese ntive fowl, ll of the hplotypes in G. g. gllus nd G. g. spdiceus from different res fell into the primry 5 clusters of Chinese ntive, which indicted the very close genetic reltionships between these two subspecies nd Chinese ntive. But the genetic distnces between Chinese ntive nd G. g. bnkiv, G. lfyettei, G. sonnerti nd G. vrius were reltively further. In ddition, Both Tble 5 nd Figure 2. showed tht the genetic distnce between G. vrius nd ntive ws not s the lrgest s hd reported (Hshiguchi et l., 1993; Cheng et l., 1996; Fumihito et l., 1996). It mybe necessry to further clrify this issue by incresing the smple size of the popultions studied. DISCUSSION Diversity nlysis of mtdna in Chinese ntive breeds Generlly, the more ncient the popultion ws, the longer they hd to mutte nd ccumulte the muttions. So ncient popultions would be more diversified geneticlly nd the hplotypes present in them would hve more opportunities to be shred by other popultions (Torroni et l., 1993; Wrd et l., 1993). The results of the present study showed tht Tibetn (ZJ), Henn cockfighting (DJ) nd Lngshn (LS) hd only one hplotype ech, but they were ll reltively ncient breeds (Qiu et l., 1988). This could be ttributed to the following resons. (i) These breeds, s germplsm, were introduced Tble 5. The verge divergence of hplotypes found in Chinese ntive fowl nd other Gllus 1 2 3 4 5 6 7 1 Ntive fowl 0.016726 2 G.g.pediceus 0.020569 0.021462 3 G.g.llus 0.022627 0.024819 0.020141 4 G.g.bnkiv 0.326823 0.327016 0.32779 0.293508 5 G.sonnerti 0.557238 0.559667 0.571293 0.419746 0.000000 6 G.fyettei 0.554068 0.558349 0.562595 0.375933 0.353075 0.010166 7 G.vrius 0.423851 0.423586 0.429228 0.42855 0.451837 0.402868 0.549169 G.sonnerti hd only one hplotype studied.

GENETIC VARIABILITY OF mtdna SEQUENCES IN CHINESE NATIVE CHICKEN BREEDS 907 0.05 bnkiv2 bnkiv1 bnkiv3 QY1 GuS8 CH6 gllus5 spdiceus7 SG2 BeH YJ6 CH5 CH2 spdiceus5 spdiceus6 WM1 spdiceus1 GuS6 DG2 BY1 SG4 BY2 ZJ1 YG3 QY4 gllus3 YG2 YG1 GuS5 gllus10 spdiceus2 spdiceus3 spdiceus8 spdiceus9 gllus4 gllus11 YJ4 QY5 gllus12 gllus13 spdiceus4 gllus2 gllus8 gllus9 YJ5 YJ2 YJ7 YJ1 GuS7 gllus6 gllus1 gllus7 sonnerti lfyett1 lfyett2 vrius1 vrius2 Figure 2. Unrooted NJ phylogenetic tree of hplotypes found in Chinese ntive fowls nd four species of Gllus. on smll scle from their primry distribution regions to new loction, the Poultry Institute of the Chinese Acdemy of Agriculturl Sciences. This process would likely result in genetic drift (founder effect). (ii) The long history of selection nd breeding or orgnized production might hve imposed high selection pressure on these ncient breeds (Pndey et l., 2002). For exmple, the cockfighting breed hd been bred for fighting for more thn 2,000 yers go. Thus, the selection pressure on this breed would be high becuse of the needs of cockfighting. (iii) These breeds were likely to hve encountered serious genetic drift (bottleneck effect) during their evolutionry process. For exmple, Henn cockfighting ws lmost extinct during the Culturl Revolution of the 1970s (Wu, 2003). (iiii) It might be cused by the smll smple sizes of the present study. Li et l. (2001) noted tht Tihe silky (SY) hd been bred for more thn 1,000 yers, nd the Annls of Ntive Livestock nd Poultry Breeds in Yunnn Province (Compiltion Committee) reported tht Chhu (CH) often mted with G. gllus inhbiting the nerby res during hrvest time. G. gllus is considered to be the originl ncestor of domestic. Our results (Tble 4) showed tht Chhu hd the most hplotypes shred with other breeds, nd the number of hplotypes in Tihe silky shred with other breeds rnked second. This suggested tht Tihe silky nd Chhu were likely to be ncient breeds or they shred ncient lineges. This ws consistent with the descriptions given in both the Annls refered to bove. Genetic differentition of chinese ntive breeds Ohno (1997) deduced tht set of full or mternl hlfsisters should hve inherited identicl mitochondril genome from their mother, but ech sister s femle descendnts invribly estblish n independent linege which in time would ccumulte its own chrcteristic muttions to become distinct sublinege. As whole, Figure 1 showed tht the phylogenetic tree of hplotypes in the Chinese ntive breeds hd five clusters which represented 5 lineges, so we concluded tht the present Chinese ntive breeds likely shred 5 common mternl lineges. As illustrted in both Figure 1 nd Tble 4, some breeds hd more thn one hplotype, nd some of these hplotypes belonged to different lineges. For exmple, some hplotypes in Gushi (GuS) belonged to lineges I, II, IV nd V. Some hplotypes in Ynjin Wugu (YJ) belonged to lineges I, II, III nd IV. Some hplotypes in Tihe silky (SY) belonged to lineges I, II nd III. Some hplotypes in Qingyun blotted (QY) belonged to lineges I, III nd V, nd some hplotypes in Chhu (CH) belonged to lineges I, II nd V. All of the bove suggested tht these breeds shred lineges, tht their genetic bckgrounds were complex, nd tht the 5 lineges might not hve evolved independently. Mting might hve occurred between lineges or some of them might hve differentited during the process of evolution. Figure 1 lso showed tht ech linege contined more thn one breed. Lineges I nd II contined most of breeds we exmined in the study, they might be the primry

908 LIU ET AL. evolutionry lineges. Tble 4 lso illustrted tht severl breeds shred the sme hplotype. Both results suggested tht these breeds belonged to the sme linege or tht they shred the common mternl ncestor regrdless of externl fetures nd ecologicl types of these breeds. How could some breeds with different fetures shre common linege? Zhu (1958) suggested tht muttion ws the min reson nd tht the more thn 30 subspecies or vrints of domestic were formed by grdul muttions. For exmple, in 1921, Joes discovered Leghorn with fuzz which ws similr to silky. Then Joes mted the strnge Leghorn with norml Leghorn nd silky, respectively, nd nlyzed the inheritnce nd differentition of the fether in their offspring. It ws found tht both the Leghorn with fuzz nd silky hd essentilly the sme muttion. But how could different breeds shre the sme mternl linege? Chinese reserchers presumed tht the primry reson for domestiction of ws to mke vilble source of met nd for religious purposes. Lter on the cockfighting breed ws bred for recretion nd lstly, egg type breeds were developed (Cheng et l., 2000). But foreign reserchers speculted tht domesticted ws first used s recretionl breeds, such s gmecock, then for vrious religious purposes, nd eventully s the source of met nd egg (Mson, 1987). The differences between these two viewpoints were ttributed to different cittions. The former minly referred to Chinese rcheologicl nd other relted documents, while the ltter minly referred to Indin rcheologicl nd other relted documents. Although the viewpoints were not consistent with ech other, both of them implied tht vrious types of breeds might hve originted from common ncestors. Originl ncestor of Chinese ntive breeds nd originl domestiction site of ntive In so fr s originl ncestor of the ntive fowl, some studies before hve probed into it using methods such s biochemicl genetics nd nucler DNA (Hshiguchi et l., 1993; Cheng et l., 1996; Mohd-Azmi et l., 2000). All of them concluded tht the domestic from different res or countries ws geneticlly very close to their indigenous red jungle fowl (G. gllus). Our results (both Tble 5 nd Figure 2) showed tht Chinese ntive breeds showed low genetic reltionship with G. vrius, G. lfyettei nd G. sonnerti. For G. gllus, however, the Chinese ntive fowl ws just geneticlly close to two subspecies of G. gllus (i.e., G. g. gllus nd G. g. spdiceus), but nother subspecies, G. g. bnkiv ws remote from them. This is in ccordnce with the results before (Fumihito et l., 1996). Our results (Figure 2) further showed tht ll of the hplotypes in G. g. spdiceus from Thilnd, Los, Mynmr nd Yunnn province of Chin, nd in G. g. gllus from Vietnm, Thilnd, Philippines, Lombok Islnd nd n unknown smpling re cn be found in the primry 5 hplotype clusters of Chinese ntive. For ech linege, the men rtes of bse substitutions were equivlent (Zheng, 1995). Therefore, the current ntive nd jungle fowls cn respectively be ssimilted to domestic nd jungle fowls t the beginning of domestiction. It is concluded tht Chinese ntive breeds originted from two subspecies of G. gllus, nmely G. g. gllus nd G. g. spdiceus. While nother subspecies, i.e., G. g. jbouillei ws not included in this study. Fumihito et l. (1996) reported tht the sequence divergence mong D-loop segments of domestic breeds nd G. g. gllus in Thilnd ws only 0.5 to 3.0%, nd hplotypes of domestic nd G. gllus from Asin popultions including G. g. gllus nd G. g. spdiceus from Thilnd nd its djcent regions were clustered in the sme group. Thus suggested tht the ll domestic breeds were likely to hve originted from single domestiction event in Thilnd nd its djcent regions, nd these domesticted then dispersed northwrds to Chin in ccordnce with the findings of West nd Zhou (1988). However, different viewpoint ws proposed due to the following resons: (i) The present distribution re of G. gllus, including ll kinds of subspecies, stretched from the northwest of Indi estwrds to the north of Chin, including Hinn province, nd southwrds to Indonesi. Moreover, G. gllus hd emigrted to the Pcific islnds (Mson, 1991). According to Fumihito et l. (1996), the distribution of G. g. gllus nd G. g. spdiceus which included Thilnd nd South Sumtr ws just prt of the distribution re of G. gllus. In the present study, except tht G. g. gllus from Sumtr ws slightly remote from ntive fowl s the result before (Fumihito et l., 1996), the genetic reltionship between Chinese ntive breeds nd G. g. gllus nd G. g. spdiceus from different res, such s Mynmr, Chin, Los, Thilnd, Vietnm, Philippines nd Lombok Islnd ws very close. (ii) Some pictures, sttues nd skeletons of were excvted in Hrp site nd Mohenjo-Doro site long the Indus Vlley. These remins were considered the most ncient nd evident proof of domestiction. The skeletons were lrger thn those of G. gllus, which ment hd been domesticted t tht time. Bsed on these remins, Zeuner speculted tht the domestiction event hd lredy been finished bout 2000 B.C., but Wood-Gush presumed tht the domestiction event hd been dted to bout 3200 B.C. (Mson, 1987). Since the founding of P. R. Chin, erly Neolithic sites were discovered in Wnnin county of Jingxi province nd Bnpo in Xi n, Shnxi province. Bones of ncient jungle fowls were found in both sites, which implied tht ncient jungle fowls hd inhbited regions long both the Hunghe nd Yngtse Vlleys.

GENETIC VARIABILITY OF mtdna SEQUENCES IN CHINESE NATIVE CHICKEN BREEDS 909 Besides those sites, bones were unerthed in other Neolithic sites in Xinzheng county of Henn province, Tengxin county of Shndong province nd Wu n county of Hebei province s well. Archeologicl nlysis of these remins suggested tht these bones were the most ncient bones dting bout 6000 to 5500 B.C. (Cheng et l., 2000). Although it ws not ble to be determined whether the bones were from domestic or jungle fowls, reserchers greed tht the bones were t lest from some species of Gllus. Thus, there should hve been similr or more ncient remins of some species of Gllus in Thilnd nd its djcent regions if the originl domestiction event hd occurred there. However, before the finding of such convincing rcheologicl evidence, it is resonble to ssume tht becuse of the suitble ecologicl environment, the present G. gllus which shre common lineges with domestic, inhbit their own distribution regions which re not necessrily the originl domestiction sites of the domestic. REFERENCES Chng, H. 1995. Conspectus of Genetic Resources of Livestock. Chinese Agriculture Press, Beijing, Chin. Cheng, G. C., D. W. Zhou nd L. C. Wu. 1991. Studies of bloodgroups of :. Anlysis of blood-groups nd plsm protein polymorphisms of eleven Chinese ntive fowl breeds. Act Genetic Sinic. 18:415-423. Cheng, G. C., K. F. Liu, Q. Zhng nd Z. X. Dn. 1996. Genetic reltionship mong domestic fowl nd G.gllus. Act Genetic Sinic. 23:96-104. Cheng, G. C., F. M. Hung, Q. X. Zhou, W. C. Bo nd Z. X. Dn. 2000. Germplsm Chrcteristics of Chinese Ntive Chicken Breeds. Shnghi Sci.-Tech. Press, Shnghi, Chin. 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